The overall aim of this proposal is to examine the events involved in the biosynthesis of calcitonin in both normal and tumor tissues. This will include a structural examination of the gene which codes for human calcitonin in an attempt to define DNA sequences important for initiation of transcription of the calcitonin gene as well as non-structural, intervening sequences which are removed during processing of calcitonin messenger RNA precursors. The calcitonin gene will be isolated from a human genomic library and sequenced by the chemical method of Maxam and Gilbert and the chain-termination method of Sanger. I will examine with sensitive cDNA hybridization assays the production of calcitonin messenger RNAs in non-thyroidal human tumors, including pheochromacytomas and small cell carcinomas of the lung, that have been associated with the ectopic production of calcitonin. These studies will be greatly facilitated by the availability of cloned rat calcitonin cDNA which cross-hybridizes with human calcitonin messenger RNA. The structure of calcitonin mRNAs expressed outside the thyroid will be determined by the synthesis, cloning and DNA sequence analysis of recombinant DNAs prepared from tumor-derived mRNAs. The regulation of expression of the calcitonin gene in both thyroid and non-thyroidal tumors where calcitonin is produced will be examined by hybridization assays which quantitate changes in amounts of calcitonin RNA in response to factors (calcium, cyclic AMP) that effect calcitonin secretion and presumably synthesis. Finally, I will examine the effects on calcitonin biosynthesis and secretion by altering the structure of the protein precursor which contains calcitonin. This will be accomplished by altering the cDNA that codes for the calcitonin precursor with restriction enzymes or the exonuclease BAL31 and inserting the altered cDNAs into specially constructed yeast or adenovirus expression vectors. The effects of these alterations on calcitonin biosynthesis and secretion will be examined in both yeast and Hela cell cultures by radioimmunoassay and limited N-terminal amino acid sequence analyses. The proposed studies should lead to a broader understanding of the basic mechanisms whereby cells synthesize and process proteins destined for secretion.